This invention relates to a device and apparatus for the simultaneous detection of multiple analytes.
Traditionally, structurally-diverse analytes have been analysed by means of specific methods, e.g., enzyme immunoassay, high performance liquid chromatography, gas chromatography, enzymatic methods and colorimetric methods. These methods are predominantly one-analyte, one-test methods.
Automation of analytical methods has generally focused on batch and random access analysers, where multiple analysis on individual test samples is performed using sequential individual test methods. This necessitates the use of multiple packs of individual test kits. In addition, analysis requires employment of several types of equipment, e.g. clinical chemistry analysers, HPLC, GCMS, automated immunoassay instruments or atomic absorption instruments.
A multi-analyte system should involve a means of providing simultaneous analysis of several analytes in a test sample. This analysis should provide results which identify individual analytes and enable the quantitation of each individual analyte in that test sample. A method of multi-analyte analysis is often claimed but the given criteria are generally not both fulfilled.
In a multi-analyte system, a typical substrate contains a plurality of individual test reaction sites each possessing a different binding ligand. The test sample contacts each of the reaction zones and thereafter a range of detection techniques is implemented to identify the analyte present. It is important that the detection method used enables quantitation of each individual analyte.
In order to produce multi-analyte arrays of spatially-distinct areas of biologically-active ligands on a substrate, the most common approach has been through photolithographic techniques. The substrate is coated with a photolabile linker. In theory, this linker should only become reactive towards a binding ligand following irradiation with light of a suitable wavelength. Spatial resolution is achieved by placing a physical mask (normally manufactured from chrome) on the substrate. The pattern of holes in the mask determines the pattern of binding regions on the substrate.
For each biological ligand to be immobilised, the general protocol is: irradiation of first sites, incubation of irradiated substrate with a first ligand to be immobilised, washing to remove loosely bound ligand, blocking unreacted sites activated by the irradiation step, and irradiation of regions where the second biological ligand is to be immobilised, with subsequent steps repeated as for the first ligand. The spatial resolution is dictated by controlling the site of irradiation, either by controlling the site of irradiation by means of a coherent UV light source from a laser or by a number of physical masks and an incoherent light source. This makes the task of immobilising a plurality of biological ligands a time-consuming process. Another disadvantage of the photolithographic approach is the need for expensive physical masks or a laser light source. Further, there is a high degree of non-specific binding.
For example, the use of arylazides, fluoro-arylazides and benzophenones has been associated with a high degree of non-specific binding. High non-specific binding results in assay background being high, significantly reducing the dynamic range of each multi-analyte assay. The non-specific binding is largely due to passive adsorption of molecules to the non-activated photolabile linker surface through ionic interactions, Van der Waals forces etc.
WO-A-9516204 describes a photolithographic approach to reducing the problems associated with high non-specific binding. In this approach, the surface linking molecule was avidin and the photolabile molecule was photobiotin or a derivative thereof. Whilst reduced non-specific binding is claimed, this technique still requires the time-consuming sequences outlined above. Immobilisation of a plurality of 20 separate biological ligands would require a total of 80 steps, assuming the basic requirement of irradiation, binding, blocking and washing steps for each separate ligand to be immobilised.
Spatial resolution has also been achieved by passive adsorption. For example, U.S. Pat. No. 5,432,099 discloses binding whereby the ligand to the substrate surface through a combination of ionic interactions, hydrophobic interactions, and Van der Waals forces.
Passive adsorption processes are dependent on changes in pH, temperature, ionic strength and on the type of substrate used, making the binding process more difficult to control.
The major drawback with this approach is the susceptibility of a proportion of weakly immobilised ligand to be desorbed during the washing step or incubation steps of the biological assay, resulting in poor intra and inter-assay precision.
A cross-linker used in many publications has been glutaraldehyde. This linker presents many disadvantages, including the tendency of proteins to cross-link which is likely to alter the function of the protein. A further disadvantage is that the coupling procedure should include a reduction step which is time-consuming and potentially very hazardous, e.g. if sodium cyanoborohydride is used as the reducing agent.
Heterobifunctional linkers have been used but in many cases these involve the need for free sulphydryl groups on the protein to be bound. This necessitates modification of the protein prior to immobilisation.
In a multi-analyte assay, it is desirable to provide both qualitative and quantitative results. Multi-analyte assays have been available for antibiotics, for example. These are largely based on microbial inhibition assays, where an antibiotic present in the sample inhibits bacterial growth and forms a zone of clearance which is proportional to the concentration of the antibiotic present in the sample. However, this method cannot provide any indication as to the identity of the antibiotic, or an accurate determination of its concentration. Microbial inhibition methods are also very slow, the complete process taking several days.
Chemical screening methods such as high performance liquid chromatography (HPLC) or gas/liquid chromatography mass spectrometry (GCMS/LCMS) struggle to accommodate the structural diversity/polarity extremes of each antibiotic group, e.g., penicillins, sulphonamides, aminoglycosides, tetracyclines etc. In addition, chromatographic methods necessitate extensive sample preparation in order that the signal-to-noise ratios are such that the required detection limits can be achieved.
Available multi-analyte devices include the Triage (see Clinical Chemistry 38(9):1678-1684 (1992)) and Advisor (see Clinical Chemistry 39(9):1899-1903 (1993)). These devices are only suitable for qualitative analysis.
The Triage device is for the simultaneous detection of a panel of seven drugs of abuse in human urine. Each device is only capable of analysing one urine sample. At the end of the procedure, the operator visually examines each of the drug-specific test zones for the presence of a red bar. All steps of the assay protocol must be performed manually by the operator. There is also no hard-copy of the test result available.
The Advisor device is similar in its application to that of the Triage device. The Advisor device screens for five different classes of drugs of abuse. The device operates using agglutination assay principles, with individual channels for each drug. All steps of the assay protocol are performed by the operator. Negative samples have agglutinated particles, whereas positive drug samples provide an unaggregated pattern of particles.
The majority of biosensors/microfabricated devices for biological application have employed silicon as the substrate. Others use glass or quartz substrates. Silicon has a very controlled crystallographic structure with well-defined crystal planes. The uniformity of the silicon substrate makes it an ideal choice for the development of a multi-analyte test device.
However, dark substrates such as silicon give so-called black body effects. In the case of detection by fluorescence, in which a fluorophore is excited using light of a particular wavelength, a dark substrate may absorb the incident excitation light energy, thus diminishing light emission from the fluorophore.
According to the present invention, a device for performing multi-analyte assays, comprises a substrate and a multiplicity of discrete reaction sites each bearing a ligand covalently bound to the substrate, and in which the surface of the substrate between the reaction sites is inert with respect to analyte. This invention thus provides a solid state, multi-analyte device which exhibits little or no non-specific binding.
A device of the invention may be prepared by activating the surface of a suitable substrate, and applying an array of ligands on to discrete sites on the surface. If desired, the other active areas may be blocked. The ligands may be applied in an aqueous system, and it is preferred if the other areas are rendered hydrophobic. The ligands may be bound to the substrate via a linker. In particular, it is preferred that the activated surface is reacted successively with an organosilane, a bifunctional linker and the ligand.
As part of this invention, bifunctional cross-linkers have been used to provide highly efficient coupling chemistries between organosilanes covalently immobilised on microfabricated silicon or ceramic substrates. Biological ligands can thus be immobilised in multi-analyte arrays.
This invention removes the need for multiple individual test reagent kits and also several instrument types, thereby facilitating the simultaneous detection of multiple analyses on a single sample. The invention provides a single integrated analyser capable of providing simultaneous detection of a wide range of chemistries. In addition, test reagents may be supplied in a combined format for a particular panel of analytes.
As part of this invention, a plurality of biological ligands is immobilised in a spatially-defined pattern of spots or lines by means of microfluidic dispensing of the ligand onto a chemically-activated substrate. The biological ligand is covalently attached to the substrate. The coupling efficiency of the biological ligand can be such that the chemical reaction is completed within a few minutes. The immobilisation procedure can ensure that the biological ligand retains its biological activity both in the short term and in the long term.
This invention also provides an integrated analyser system for the simultaneous detection of a wide range of analytes in a multi-analyte format. The analyser system is designed for maximum end-user convenience, with the capability of obtaining multi-analyte identification and quantitation from each test sample. The preferred analyser system is a combination of a X-Y translational platform, a sample handling unit, liquid handling/flow control means, a temperature-controlled dark box, a CCD camera, and image-processing software. The platform may be associated with a stepper motor, to achieve a positioned accuracy of, say, 10 xcexcm, for positioning device(s) at each stage of the analytical procedure.